Review Article. Different HPLC analysis method of itraconazole

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1 Available online Journal of Chemical and Pharmaceutical Research, 2016, 8(3):58-63 Review Article ISSN : CODEN(USA) : JCPRC5 Different HPLC analysis method of itraconazole Verma Vikrant * and Singh Umesh Kumar Department of Pharmacy, Kharvel Subharti College of Pharmacy, Swami Vivekanand Subharti University, Meerut ABSTRACT The present review article deals with the method developed for itraconazole by HPLC method. A wide variety of columns, mobile phase combinations, degassers used for the study. Different flow rates where adjusted for mobile phase combinations. In some methods forced degradation studies was also carried out and the methods developed were validated according to the ICH guidelines. After studying different research articles it came to light that HPLC method development of itraconazole has been done in different dosage forms of itraconazole and still there is huge potential for new methods to be developed with different mobile phase and column combinations. These new method data are required by regulatory agencies. Keywords: Itraconazole, Linearity, Antifungal INTRODUCTION Itraconazole is a (1-(butan-2-yl)-4-{4-[4-(4- {[2R,4S)-2-(2,4-dichlorophenyl)-2-(1H-1,2,4 triazole- 1- ylmethyl) 1, 3 dioxolan 4 -yl] methoxy}phenyl) piperazin -1-yl ] phenyl} 4, 4-dihydro-1H-1,2,4- triazole-5-one) is member of the drug class known as anti-fungal. It is used for the inhibition of fungal cytochrome p450 enzyme lanosterol 4 demethylase, used in the conversion of lanosterol to ergosterol, which is a main sterol in fungal cell membrane, thus inhibits replication and promotes cell death in case of the yeast cells transformation into hypothetically invasive hyphae. Literature survey revealed that very few methods have been reported for the analysis of Itraconazole which include UV spectroscopy, Reverse Phase High Performance Liquid Chromatography, Ultra Pressure Liquid Chromatography, LCMS, HPTLC methods. The present article deals with existing methods developed for itraconazole by HPLC. Sarvani Paruchuri et al. developed a simple, economic, selective, precise and accurate reverse phase high performance liquid chromatography method for analysis of Itraconazole and Related Substances and validated according to ICH guidelines. Itraconazole was well separated using Thermo Hypersil BDS C18, 150mm Χ 4.6 mm, 5µm column for assay quantification in isocratic mode with mobile phase comprising of buffer: Acetonitrile (65:35) with a flow rate of 1.5ml/min and Thermo Hypersil BDS C18, 100 mm x 4.6 mm, 3 µm column for Related substances quantification in gradient mode with mobile phase comprising of 0.08M tetra butyl ammonium hydrogen sulphate: Acetonitrile with a flow rate of 1.5ml/min. 58

2 The retention time was found out to be 6.2min and % assay was found to be 99.9%. The percentage recovery was found to be 99.6 to 101.2%. Proposed method was validated for precision, accuracy, linearity, range, specificity and robustness. The drug was subjected to forced degradation and stability studies. 1 Important parameter found in the study are given in Figure1,Table1-3. Figure1. Linearity parameter Table.1.Validation Parameters Parameters Assay Linearity mcg/ml Precision (%RSD) 0.2 Accuracy Assay 99.9 Ruggedness(%RSD) 0.33 Table.2.Optimised Chromatographic Conditions Parameters Assay Column Thermohypersil BDS C18 150Χ4.6mm, 5µm Mobile phase Buffer: ACN (65:35) Flow rate 1.5ml/min Wave length UV at 225nm Injection volume 10µl Column temperature 25 0 C Run Time 12min Table.3.Linearity Data Related substances Mcg/ml Area Rt M. Salomi, et al. Developed a validated RP-HPLC method for the estimation of Itraconazole in pure and pharmaceutical dosage form. In this a simple, fast and precise RP HPLC method was developed for the 59

3 quantification of Itraconazole in pure and pharmaceutical dosage form. The quantification was carried out using Dionex C X 250mm, 5µm enhanced polar selectivity column and mobile phase comprised of methanol and ph 7.5 potassium dihydrogen phosphate in the ratio of 40:60 and degassed under ultrasonication. The flow rate was 1.5ml/min and the effluent was monitored at 306nm. The retention time of Itraconazole was found to be 5.2 min. The method was validated in terms of linearity, precision, accuracy, specificity, robustness, limit of detection and limit of quantitation in accordance with ICH guidelines. Linearity of Itraconazole was in the range of µg/ml. The percentage recoveries of Itraconazole were 99.33% to 99.66% from the capsule formulation. The proposed method is suitable for determination of Itraconazole in pharmaceutical dosage form. 2 Important parameter found in the study are given in Table4-6. Table 4: Optimized chromatographic parameters Optimized Chromatographic parameters Elution Isocratic Mobile phase methanol and ph 7.5 KH 2PO 4 (40:50) Column Dionex C18column Flow rate 1.5ml/min Detection 306nm Injection volume 10µl Retention time min Run time 7 min Table 5: System suitability parameters Parameters Values Retention time min Theoretical plates Tailing factor Table 6: Linearity results for Itraconazole S.No Concentration (µg/ml) Area AU (n=6) Kumudhavalli et al., developed a simple, specific, accurate and precise reverse phase high performance liquid chromatographic method and validated for the estimation of Itraconazole in capsule dosage form. An inertsil C-18, 5µm column having 250 x 4.6mm internal diameter in isocratic mode with mobile phase containing Tetrabutyl ammonium hydrogen sulphate buffer solution and Acetonitrile in the ratio of 40:60v/v was used. The flow rate was 1.5ml/min and effluents were monitored at 225nm. The retention time for Itraconazole was 5.617min. The method was validated for linearity, accuracy, precision, specificity, limit of detection, limit of quantification and robustness. Limit of detection and limit of quantification were found to be 0.85µg/ml and 2.60µg/ml respectively and recovery of Itraconazole from capsule formulation was found to be 98.3 to 100.3%.The system suitability parameters such as theoretical plates and tailing factor were found to be and 1.08 The proposed method was successfully applied for the quantitative determination of Itraconazole in capsule formulation. 3 Important parameters are given in table 7 and table 8 Table 7: Validation and System Suitability Parameters Linearity range (µg/ml) µg/ml Correlation coefficient Retention time (min) 5.61 Tailing factor 1.08 Limit of detection (µg/ml) 0.85 µg/ml Limit of quantification (µg/ml) 2.60 µg/m Precision (RSD %) 0.27% 60

4 Table 8: Result of Robustness Study Factor Level Mean* %RSD Flow rate (ml/min) 1.4ml/min ml/min Mobile phase ratio 8: : Column temperature o C Azevedo et al. developed HPLC-FLD Method for Itraconazole Quantification in Poly Lactic-co-glycolic Acid Nanoparticles, Plasma and Tissue. In this a high performance liquid chromatography (HPLC) procedure using fluorometric detection was developed for determination of itraconazole in polymeric poly(lactic-co-glycolic acid) nanoparticles, plasma and tissue. Linearity, limits of detection and quantification, recovery, precision, selectivity and stability were established. The developed method was tested in itraconazole detection and quantification of biodistribution of nanoparticles administered intraperitoneally to Balb/C female mice. This study developed an analytical method for HPLC with fluorometric detection for quantification of itraconazole in polymeric nanoparticles, tissue and plasma, which is sensitive, low cost, viable for routine usage and with potential for application in itraconazole biodistribution and pharmacokinetics studies. 4 Using the developed analytical method, ITZ retention time was 7.6 min and internal standard retention time was 9.1 min with 11 min for each run. The chromatographic runs were completed at 11 min, a short run when compared to most methods for ITZ quantification in HPLC, resulting in less waste, in accordance with the principles of green analytical chemistry. Important parameters found are given in table 9 Table 9 Linear regression parameter and limits of detection and quantification for itraconazole (area vs. Concentration) Calibration curves Range of concentration / LOD / LOQ / Slope Intercept r2 (µg ml 1) (µg ml 1) (µg ml 1) Mobile phase Mobile phase Kideny Liver Lung Spleen Plasma Andelija Malenovic et al. Chemometrically assisted optimized and validated an RP-HPLC method for the analysis of itraconazole and its impurities. In this chemometrically assisted optimization and validation of the RP-HPLC method intended for the quantitative analysis of itraconazole and its impurities in pharmaceutical dosage forms was carried out. To reach the desired chromatographic resolution with a limited number of experiments in a minimum amount of time, Box Behnken design was used to simultaneously optimize some important chromatographic parameters, such as the acetonitrile content in the mobile phase, ph of the aqueous phase and the column temperature. Separation between itraconazole and impurity F was identified as critical and selected as a response during the optimization. The set optimal mobile phase composition was acetonitrile/water ph 2.5 adjusted with o-phosphoric acid (50:50, V/V). Separations were performed on a Zorbax Eclipse XDB-C18, mm, 5 m particle size column with the flow rate 1 ml min 1, column temperature set at 30 o C and UV detection at 256 nm. The established method was then subjected to method validation and the required validation parameters were tested. 5 Important parameters found are given in table 10 and table 11 61

5 Table10. Important parameters for linearity Parameter Itraconazole Impurity B Impurity F a b R t b Table 11. Important parameters for accuracy Concentration 1 (µg ml 1 ) Recovery 1 (%) Concentration 2 (µg ml 1 ) Recovery 2 (%) Concentration 3 (µg ml 1 ) Recovery 3 (%) Durisehvar Ozer Unal et al. developed method for the determination of ITR and metabolite from plasma by LC- MS/MS system, which is reliable and robust. The method uses simple and economical liquid-liquid extraction of the drugs from human plasma. The lowest standard 1.0 ng/ml for ITR and 2.0 ng/ml for HITR on the calibration curve was identified as the lower limit of quantification (LOQ) with a precision of less than or equal to 20 %. The calibration curve was prepared from seven spiked plasma samples within the range of ng/ml, including LOQ for ITR and ng/ml, including LOQ for HITR. The standard calibration curves was linear over the concentration range from ng/ml with mean r 2 = for ITR and r 2 = for HITR respectively. The LOQ was 1.0 ng/ml for ITR and 2.0 ng/ml for HITR. The calibration curve had a regression equation of y= x for ITR, y= x and for HITR respectively. The limit of detection of developed method is as low as LC-MS/MS system by liquid-liquid extraction procedure. The validation results were included specificity, accuracy, extraction recovery, linearity and range. The assay can be applied easily successfully to the pharmacokinetic and bioequivalence studies. 7 Important parameters found are given in table Table 12 intra-day coefficient of variation and relative error% for determination of HITR Sample Conc. of HITR ( ng/ml) Mean conc. of HITR (ng/ml) Relative Error% SD CV% n QC QC QC QC QC Table 13. Intra-day coefficient of variation and relative error % for determination of ITR Sample Conc. of ITR ( ng/ml) Mean conc. of ITR (ng/ ml) Relative Error% SD CV% n QC QC QC QC QC Table 14. The Intra And Inter Day CV% Value Itraconazole Hydoxy-ITR (1-600 ng/ml) (2-600 ng/ml) Min. Max. Min. Max. Inter-day CV% Range(n=6) Intra-day CV% Range(n=18)

6 REFERENCES [1] Sarvani Paruchuri, Haritha Pavani K ; Indian Journal of Research in Pharmacy and Biotechnology November December 2013 Page [2] B. Thangabalan, M. Salomi, N. Sunitha, S. Manohar Babu ; Asian J. Pharm. Ana. 2013; Vol. 3: Issue 4, Pg [3] V. Kumudhavalli ; Py-Daniel, Osmindo R. Pires Junior, Carlos M. Infante C., Maria L. Fascineli,Antonio C. Tedescod and Ricardo B. Azevedo, J. Braz. Chem. Soc., Vol. 00, No. 00, 1-7, [4] Karen R. Py-Daniel, Osmindo R. Pires Junior, Carlos M. Infante C., Maria L. Fascineli,Antonio C. Tedescod and Ricardo B. Azevedo, HPLC-FLD Method for Itraconazole Quantification in Poly Lactic-co-glycolic Acid Nanoparticles, Plasma and Tissue, J. Braz. Chem. Soc., Vol. 00, No. 00, 1-7, 2014 [5] Irena Kasagic Vujanovic, Andelija Malenovic,Marko Jovanovic, Tijana Rakic,Biljana Jancic Stojanovic,Darko Ivanovic, Acta Pharm. 63 (2013) [6] Zeynep Irem Diler, Durisehvar Ozer Unal, Dilek Demir Erol, Hacettepe University Journal of the Faculty of Pharmacy,Volume 30 / Number 2 / July 2010 / pp

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